Single-Neuron Transcriptome and Methylome Sequencing for Epigenomic Analysis of Aging
Open accessAbstract
Enormous heterogeneity in transcription and signaling is the feature that slows down progress in our understanding of the mechanisms of normal aging and age-related diseases. This is critical for neurobiology of aging where the enormous diversity of neuronal populations presents a significant challenge in experimental design. Here, we introduce Aplysia as a model for genomic analysis of aging at the single-cell level and provide protocols for integrated transcriptome and methylome profiling of individually identified neurons during the aging process. These single-cell RNA-seq and DNA methylation assays (methyl-capture/methyl enrichment) are compatible with all major next generation sequencing platforms (we used Roche/454 and SOLiD/Life Technologies as illustrative examples) and can be used to integrate an epigenetic signature with transcriptional output. The described sequencing library construction protocol provides both quantitative and directional information from transcriptional profiling of individual cells. Our results also confirm that different copies of DNA in polyploid Aplysia neurons behave similarly with respect to their DNA methylation.
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A reduced representation sequencing protocol for 454 and Ion Torrent platforms can detect low abundance transcripts, reduces the amount of sequencing, and gives more accurate quantification.
“We developed a reduced representation sequencing protocol for the 454 and Ion Torrent sequencing platforms that can detect low abundance transcripts 47 . The method reduces the amount of sequencing and gives more accurate quantification and additional details of the procedure are reported elsewhere 47,48 .”
From The ctenophore genome and the evolutionary origins of neural systems (2014) · cites this paper as [48]
Methods §4.3 describes a single-neuron RNA-seq library protocol for 454 sequencing, and §4.4 says RNA-seq can detect very low-abundance RNAs and describes validation of quantification. The copy does not establish that this protocol is for Ion Torrent, reduces the amount of sequencing, or gives more accurate quantification than an alternative; §4.4 instead associates low-abundance detection with high sampling. Page numbers are unavailable in our copy. (LLM, conf=0.85)
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- [47]Moroz LL Kohn AB 2010 Do different neurons age differently? Direct genome-wide analysis of aging in single identified cholinergic neurons Front Aging Neurosci 2 6 10.3389/neuro.24.006.2010 20725513 PMC2910937 · DOI
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The ctenophore genome and the evolutionary origins of neural systems
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Single-Cell Semiconductor Sequencing
Kohn, A. B., Moroz, T. P., Barnes, J. P., Netherton, M., & Moroz, L. L. (2013) · Methods in Molecular Biology · doi:10.1007/978-1-62703-556-9_18 · Cited by 22 · see citing works
Self-citation
- Authors
- Moroz, L. L., & Kohn, A. B.
- Affiliations
- University of Florida (US)
- Venue
- Methods in Molecular Biology
- Publisher
- Humana Press
- Published
- 2013
- Volume
- 1048
- Pages
- 323-352
- ISSN
- 1064-3745 (ISSN-L)
- 1940-6029
- PMC
- PMC4045448
- OpenAlex
- W1918583492
- Cited by
- 42
- References
- 60
- Citations by year
- 2024: 3 · 2023: 2 · 2021: 4 · 2020: 5 · 2019: 8 · 2018: 5 · 2017: 7 · 2016: 2 · 2015: 1 · 2014: 3 · 2013: 2
- FWCI
- 3.50
- Type
- book chapter
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Licence
- Locations
- Methods in molecular biology (publishedVersion)
- PubMed (publishedVersion)
- PubMed Central (acceptedVersion)
- Funders
- National Institutes of Health
- National Institute of Mental Health (R01MH097062)
- National Institute on Drug Abuse (R21DA030118)
- National Institute of General Medical Sciences (R01GM097502)
- National Center for Research Resources (R21RR025699)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Single-cell and spatial transcriptomics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Epigenetics and DNA Methylation
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Aging › Genetics, Aging, and Longevity in Model Organisms
- Keywords
- aging, Aplysia, DNA methylation, single-cell RNA sequencing, neuronal heterogeneity, polyploidy, epigenomics, age-related diseases, sequencing library construction
- MeSH terms
- Aging, Animals, Aplysia, Base Sequence, Neurons, Sequence Analysis, RNA, DNA Methylation, Gene Expression Profiling, Epigenomics, High-Throughput Nucleotide Sequencing, Transcriptome
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